Stabilization of biological fluids by addition of sterol esters

ABSTRACT

The present invention relates to the use of sterol esters for the long-term stabilization of biological fluids, in particular even those which are obtained by lyophilization and subsequent reconstitution.

[0001] The present invention relates to the use of sterol esters for thelong-term stabilization of biological fluids, in particular even thosewhich are obtained by lyophilization and subsequent reconstitution.

[0002] In all studies with biological fluids, in particular in those onwhich or with the aid of which investigations which are to be assessedoptically are to be carried out, the problem of turbidity poses itself.The cause of this turbidity is not always detectable, often it isprecipitation of protein or lipid from the solution. Additionally heldresponsible for these instabilities are the lipoprotein macromoleculesoccurring in body fluids, which on account of their content ofwater-insoluble phospholipids have a tendency to aggregate per se.

[0003] If labile protein solutions are stored, the development ofturbidity, or adsorption of the proteins on vessel walls can likewiseoccur, which considerably impairs the quality of these products.

[0004] The abovementioned instabilities are additionally increased ifthe biological fluids are lyophilized for stabilization. Examples ofbiological fluids are serum, plasma, cerebrospinal fluid, pleuralexudates or ascites of human or animal origin.

[0005] Within the meaning of the invention, such biological fluids canalso be solutions of synthetic composition formed from an artificial oralternatively natural liquid matrix (e.g. serum or phosphate-bufferedNaCl solution) known per se to the person skilled in the art and addedbiological substances, which for their part can be prepared by geneticengineering.

[0006] Such solutions of synthetic composition are often used as controlor standard sera.

[0007] Control sera are understood as meaning sera of human or animalorigin having an optionally modified, but serum-like composition, whichcontain serum constituents, for example proteins, enzymes, enzymaticallydeterminable substrates and electrolytes in a known concentration andare suitable for the control of determination methods for these serumconstituents.

[0008] In order to guarantee the shelf life of labile components suchas, for example, enzymes or lipoproteins, biological fluids can bestored in lyophilized form and/or at low temperature, preferentiallybelow −18° C. Undesired side effects of lyophilization are turbiditieswhich occur after reconstitution of the control sera due to alterationof the solubility behavior, especially of the lipoproteins. Theseturbidities often interfere in spectrophotometric methods so that, forexample, a sample blank value is additionally necessary.

[0009] Since problems often occur due to turbidity, numerous attempts atelimination have also already been undertaken. Even if solutions havealready been found, these, however, were until now essentiallyrestricted to closely defined application areas and conditions.

[0010] DE-P 31 07 060 describes the addition of organic non-sugar-likesubstances such as methanol, alanine, triethylene glycol, valine,acetate, lactate or sodium 2-hydroxymethylbutyrate. Such an addition canresult, e.g. in the case of alanine and methylbutyrate, in disturbancesof enzyme reactions.

[0011] Addition of methanol is generally injurious to health. If sodiumacetate is used, the control serum can no longer be employed as auniversal control serum for electrolyte determinations. Addition ofammonium compounds interferes with urea determinations. Other substancescan cause general test disturbances.

[0012] A further process for the avoidance of turbidities by addition ofproline and Na desoxycholate has previously been described in DE-A 33 29952. This addition, however, has the disadvantage that it leads, forexample, to artefacts during protein separation and makes difficultdeproteination with trichloroacetic acid, e.g. in the determination ofcreatinine.

[0013] The present invention was therefore based on the object offinding a process which can be generally used for the long-termstabilization of biological fluids.

[0014] Surprisingly, it was possible by addition of sterol esters tobiological body fluids to achieve a stabilization even in the presenceof lipoproteins. Even after lyophilization of treatedlipoprotein-containing body fluids with sterol esters, a clear productis obtained after dissolution.

[0015] Surprisingly, it was also found that labile protein solutions,which tend to become turbid or whose proteins adsorb easily on vesselwalls, are stabilized by addition of sterol esters.

[0016] Surprisingly, it has furthermore been found that lower turbidity,more homogeneous reconstituted control sera are prepared in particularif sterol ester is added to the control serum before lyophilization. Asa result, the serum is on the whole not only more homogeneous but alsothe precision of concentration and activity determinations of theparameters contained in the control serum is improved, i.e. theregaining of the declared theoretical values is facilitated for the userof quality control sera.

[0017] Sterol esters belong to the steroids class of substances (gonanderivatives) which generally identifies a hydroxyl group in the 3 βposition. Significant differences exist in the side chain attached inthe 17(20) position. Sterols are a large class (BEYER et al. (1981),Lehrbuch der organischen Chemie [Textbook of Organic Chemistry], pp.649-664 “Steroids”) . The experiments carried out with differentrepresentatives of this class (e.g. vitamin D3, estrone, cholesterol andstigmasterol) show that general applicability is guaranteed. Thederivatives of cholesterol are particularly advantageous.

[0018] The sterol esters according to the invention moreover have apolyethylene glycol group coupled via a dicarboxylic acid. In principle,all known dicarboxylic acids can be used, since the bifunctionalreactivity is essentially decisive for the function according to theinvention. The following dicarboxylic acids can advantageously be used:succinic acid, adipic acid, sebacic acid.

[0019] The PEG group should fundamentally also guarantee the solubilityof the sterol ester, so that the person skilled in the art, ifappropriate by means of an experiment, can easily determine the optimumlength. According to experience, the following chain lengths areadvantageous: polyethylene glycol 600, polyethylene glycol 900 orpolyethylene glycol 3000.

[0020] The turbidity behavior measured in a nephelometric measuringprocess advantageously serves as a measure of the stabilizing effect.For the person skilled in the art, it is easy with the aid of thepresent invention to employ another, corresponding process.

[0021] The invention thus relates to a process for the stabilization ofbiological fluids and the fluids stabilized in this way.

[0022] Preferred here is the use, for the long-term stabilization ofbiological fluids, of sterol esters of the general formula I

R₁—O— (O) C—R₂—C(O)—O—[CH₂—CH₂—O]_(n)—CH₂—CH₂—OH   Formula I

[0023] in which n=1-200 and

[0024] R₁=sterol

[0025] R₂=aliphatic or aromatic ring having 4 to 8 C atoms, of which atleast one can be replaced by N, S or O, or is a linear or branched chainhaving 0 to 12 C atoms, particularly preferred is use of the sterolesters in which R₁ is a compound of the general formula II:

[0026] R₄ and R₅ can be H or —CH₃

[0027] R₆ can be a straight-chain or branched chain having 1 to 12 Catoms, an —OH or ═O group,

[0028] the rings A, B, C and D can each be saturated, unsaturated oraromatic per se

[0029] and, if R₄=—C(19)H₃, the ring B between C(9) and C(10) can beopened with formation of a double bond between C(9) and C(19).

[0030] Very particularly preferred is the use of sterol esters, thesterol radical originating from cholesterol, vitamin D3, stigmasterol orestrone.

[0031] Advantageously, the sterol ester is added in such a concentrationthat the concentration in the biological fluid is 0.05-5% by weight,preferentially 0.1-3% by weight, particularly preferentially 0.5-1.5% byweight.

[0032] Preferentially, the biological fluid is lyophilized according toa process known per se to the person skilled in the art andreconstituted for use.

[0033] The biological fluid can also be frozen until use, advantageouslystored at ≦−18° C.

[0034] The invention also relates to a reagent, essentially consistingof a natural or artificial matrix, at least one diagnostically relevantsubstance (analyte) and a sterol ester of the general formula I in aconcentration of 0.05 to 5% by weight.

[0035] To obtain the effect according to the invention, the sterolesters according to the invention can also be combined with the knownstabilizers, in particular with detergents.

[0036] Particularly advantageous here are combinations of sterol esterswith nonionic and/or zwitterionic detergents. The sterol esterconcentration in such combinations can advantageously be lowered.

[0037] Biological fluids within the meaning of this invention havealready been described further above.

[0038] The concept of long-term stability is also dependent, inter alia,on the measuring process and the analyte. Fundamentally, the stability,however, should be at least 6 months, preferentially at least 12 months,particularly preferentially at least 18 months. A biological fluid isdescribed as stable within the meaning of the present invention if theturbidity measured at 546 nm has increased by no more than 300% comparedwith the turbidity of the starting fluid.

[0039] The following examples illustrate the invention withoutrestricting it.

EXAMPLE 1

[0040] Citrate plasma from a healthy donor was treated with variousconcentrations of cholesterol-PEG 900 (FLUKA CH-9471 Buchs/Switzerland,Item No. 26735) (CP) and then lyophilized according to processes knownper se to the person skilled in the art.

[0041] For documentation of the turbidity, after reconstitution of thelyophilizate the blank value was measured on a Behring nephelometer(BNA, Dade Behring Marburg GmbH, D-35001 Marburg/Germany) and theextinction was measured at 546 nm. Extinction Blank value BNA 546 nm inbit Starting 0.131 11 material: Citrate plasma (without 1.151 >3500addition) +0.2% CP 0.643 3257 +0.5% CP 0.643 683 +1% CP 0.165 84

EXAMPLE 2

[0042] Human serum was treated with various concentrations ofcholesterol-PEG 900 (CP) and stored at various temperatures (2-8° C.,−20° C.) or lyophilized.

[0043] For documentation of the turbidity, the blank value was measuredon a Behring nephelometer (BNA) and the extinction was measured at 546nm. Human serum, untreated Human serum + 1% CP Blank Blank Extinctionvalue BNA Extinction value BNA 546 nm in bit 546 nm in bit Startingvalues 0.131    98 0.130  98 4 days at 2-8° C. 0.215    952 0.152  132 4days −20° C. 0.235    581 0.171  155 1 year at −20° C. 0.790 >3500 0.2791837 Lyophilizate 1.630 >3500 0.355 1676 (stored at 2-8° C. for 1 year)after reconstitution

1. The use, for the long-term stabilization of biological fluids, ofsterol esters of the general formula I R₁—O—(O) C—R₂—C(O)—O—[CH₂—CH₂—O]_(n)—CH₂—CH₂—OH   Formula I in which n=1-200 andR₁=sterol R₂=aliphatic or aromatic ring having 4 to 8 C atoms, of whichat least one can be replaced by N, S or O, or is a linear or branchedchain having 0 to 12 C atoms.
 2. The use as claimed in claim 1 , whereR₁ is a compound of the general formula II:

R₄ and R₅ can be H or —CH₃ R₆ can be a straight-chain or branched chainhaving 1 to 12 c atoms, an —OH or ═O group, the rings a, b, c and d caneach be saturated, unsaturated or aromatic per se and, if R₄=—C(19)H₃,the ring B between C(9) and C(10) can be opened with formation of adouble bond between C(9) and C(19).
 3. The use as claimed in claim 1 ,where R₁ is selected from the group consisting of: cholesterol, vitaminD3, stigmasterol and estrone.
 4. The use as claimed in claim 1 , whereinthe sterol ester is added in such a concentration that the concentrationin the biological fluid is 0.05% by weight to 5% by weight.
 5. The useas claimed in one of claims 1 to 4 , wherein the biological fluid islyophilized and reconstituted for use.
 6. The use as claimed in one ofclaims 1 to 4 , wherein the biological fluid is frozen until use,advantageously stored at ≦−18° C.
 7. A reagent, essentially consistingof a natural or artificial matrix, at least one diagnostically relevantsubstance (analyte) and a sterol ester of the general formula I in aconcentration of 0.05 to 5% by weight.